Epitope-tagging of the endogenous murine BiP/GRP78/Hspa5locus allows direct analysis of the BiP interactome and protein misfoldingin vivo

Abstract

BiP/GRP78, encoded by the Hspa5 gene, is the major HSP70 family member in the endoplasmic reticulum (ER) lumen, and controls ER protein folding. BiP's essential functions in facilitating proper protein folding are mainly mediated through its dynamic interaction with unfolded or misfolded client proteins, and by serving as a negative regulator of the Unfolded Protein Response. A mechanistic understanding of the dynamics of BiP interaction with its protein partners is essential to understand ER biology, and therefore, we have sought to develop a tractable model to study misfolded protein interaction with BiP. For this purpose, we have used homologous recombination to insert a 3xFLAG epitope tag into the endogenous murine Hspa5 gene, just upstream from the essential KDEL signal necessary for ER localization of BiP. Tagging BiP in this way did not alter Hspa5 expression under basal or ER-stress induced conditions in hepatocytes ex vivo or in fibroblasts. Furthermore, the tag did not alter the cellular localization of BiP or its functionality. All of these findings in primary tissue culture were also confirmed in vivo in livers of heterozygous mice with one WT and one FLAG-tagged Hspa5 allele. Hepatocyte-specific BiP-FLAG modification did not alter liver function or UPR signaling. Importantly, immunoprecipitation with anti-FLAG antibody completely pulled down FLAG-tagged BiP from lysates of BiP-FLAG expressing livers. In summary, we generated a novel model that can be used to investigate the BiP interactome in vivo under physiological and pathophysiological conditions in a cell type-specific manner. This tool has the capability, for the first time, to provide an unbiased approach to identify unfolded and misfolded BiP-client proteins, and to provide new information on the role of BiP in many essential ER processes.

Ectopic FVIII expression and misfolding in hepatocytes as a cause for hepatocellular carcinoma

“…Hence, we created a genetically modified mice with a 3x FLAG tag in the endogenous BiP/GRP78/ Hspa5 locus ( BiP-Flag mice) to allow for quantitative isolation of BiP-client protein complexes using anti-FLAG magnetic beads without altering BiP function ( Figs. 4A-B ; (39, 40). Importantly, the hepatocyte-specific expression of BiP-Flag is at the same level as endogenous and is regulated identically to the endogenous Hspa5 locus.…”

“…However, there are no available antibodies that can quantitatively co-immunoprecipitate (IP) BiP-client protein complexes. Hence, we created a genetically modified mouse with a 3x FLAG tag inserted into the endogenous BiP/GRP78/ Hspa5 locus ( BiP-Flag mice) for quantitative isolation of BiP-client protein complexes using anti-FLAG magnetic beads, 39 Tagging BiP with 3X FLAG in this manner did not alter BiP function or BiP expression, which is essential to measure physiological interactions (Figs. 4A-B , 39,40 ) .…”

“…2). Using our recently generated BiP/GRP78/Hspa5-knockin mice harboring a 3X FLAG tag in the C-terminus of BiP in the endogenous BiP/GRP78/Hspa5 locus (46) to assess the folding status of BDD accumulated in hepatocytes in vivo, we show that nearly half of intrahepatocellular BDD binds BiP (Fig. 3), indicative of misfolding.…”

“…2). Using our recently generated BiP/GRP78/Hspa5 -knockin mice harboring a 3X Flag tag in the C-terminus of BiP in the endogenous BiP/GRP78/Hspa5 locus (40) to assess the folding status of BDD accumulated in hepatocytes in vivo , we show that nearly half of intrahepatocellular BDD binds BiP (Figure 3), indicative of misfolding. This finding provides evidence that supports our hypothesis that compared to N6, ectopic expression of BDD induces a much higher level of misfolded FVIII in the liver, which can induce a series of cytotoxic responses, including activation of the UPR, and ultimately leading to initiation of HCC development.…”

“…Mice were fed a regular mouse chow diet. Induction of BiP-Flag expression was initiated at the age of 6 wks by transduction with AAV8-TGB-Cre (46). AAV8-TGB-Cre-transdcued wild type littermates were used as controls.…”